Assessing Integrity of Plant RNA with the
Agilent 2100 Bioanalyzer System
Application Note
Genomics
Authors
Abstract
Suresh Babu C.V.
High quality RNA, free of genomic DNA is a critical determinant for the success of
Agilent Technologies, Inc.
many downstream techniques used in functional genomics, such as RT-PCR and
Bangalore, India
microarray-based experiments. The Agilent 2100 Bioanalyzer System and its unique
RNA Integrity Number (RIN) algorithm have become the industry standard for quality
Marcus Gassmann
analysis of total RNA because of the capability of providing sample and
Agilent Technologies, Inc.
user-independent assessment of RNA quality from minimal sample amounts.
Waldbronn, Germany
This Application Note demonstrates the use of a dedicated Plant RNA assay with the
Agilent 2100 Bioanalyzer System to assess the integrity of plant RNA samples from
multiple plant sources and differing degradative states.
Introduction
Plant tissues are comprised of three types of rRNAs – cytosolic, chloroplastic,
and mitochondrial – which vary in size from 5S to 25S (Table 11). Green tissue may
contain additional rRNAs in contrast to non-green tissues, such as roots. A dedicated method to determine plant RIN values was introduced in the B.02.07 version
of the Agilent 2100 Expert software allowing reliable RIN values to be determined
from these complex types of plant tissues. This assay was validated with 454 plant
samples to improve the detection of appropriate plant RNA peaks for RIN determination. We describe the use of this assay to provide easy, user-independent assessment of plant RNA quality with as little as picogram to nanogram amounts of RNA 2.
Materials and Methods
RNA samples
Total RNA (Plant Normal Tissue) samples were purchased from BioChain
(Hayward, CA, USA) and from the
University of Hohenheim (Stuttgart,
Germany). Water (cell culture tested)
and all other chemicals were obtained
from Sigma (St. Louis, MO, USA).
Instrument and kits
Separations were performed on the
Agilent 2100 Bioanalyzer System,
controlled by the Agilent 2100 Expert
software, using the Plant RNA Nano
assay in accordance with the RNA
6000 Nano kit protocol (Agilent
Technologies, Waldbronn, Germany).
Data analysis was performed on the
2100 Expert software, version B.02.07
25S, 8S, 5S
18S
Larger subunit
23S, 5S
Smaller subunit
16S
Smaller subunit
18S, 5S
15
18S
Marker
10
5
90
25S
80
Lettuce
70
RIN = 7.8
60
18S
50
40
30
20 Marker
10
0
_10
18 23 28 33 38 43 48 53 58 63 68
Time (sec)
Fluorescence
24S, 5S
20
Sunflower
(root)
RIN = 8.4
0
_5
18 23 28 33 38 43 48 53 58 63 68
Time (sec)
Mitochondrial ribosomes
Larger subunit
25S
25
40
35
30
25
20
25S
18S
Fluorescence
30
Chloroplasts ribosomes
Table 1
Plant ribosomal RNA.
RIN values of various plant RNA samples across different chips on the same
(intra-) and different (inter-) days show
high concordance (Table 2). All RNA
samples had a RIN value above 6.4.
The coefficients of variation (CV) of the
RIN measurements were in the range
of 1.54% to 5.46%, illustrating the
assay precision of the RNA integrity
measurements.
Sunflower
(leaf)
RIN = 6.6
15 Marker
10
5
0
_5
18 23 28 33 38 43 48 53 58 63 68
Time (sec)
80
70
60
Fluorescence
Smaller subunit
Plant RNA assay
The utility of the Agilent 2100
Bioanalyzer System Plant RNA assay
was demonstrated with multiple commercially available plant total RNA
samples. Figure 1 illustrates typical
electropherograms from sunflower
root, sunflower leaf, lettuce, and
potato RNA samples, with respective
RINs. In all samples, the abundant
25S and 18S rRNA peaks are well
resolved and automatically identified
by the software. Compared to the root
samples, the leaf and lettuce extracts
exhibit additional fast migrating peaks
corresponding to smaller chloroplast
ribosomal RNAs showing that total
RNA profiles can vary depending on
species and tissue types.
Types of RNA
Cytosolic ribosomes
Larger subunit
Results and Discussion
Fluorescence
Ribosomal RNA
Degradation study
Tobacco tissue homogenates were
incubated at room temperature for 0, 5,
10, 20, and 40 minutes followed by centrifugation to remove insoluble compounds. RNA was extracted using suitable solvents and subjected to analysis
on the Agilent 2100 Bioanalyzer System
for degree of degradation. Degradation
and extractions were performed by
Jens Göpfert at the University of
Hohenheim (Stuttgart, Germany).
50
25S
18S
Potato
RIN = 7.6
40
30
20
10 Marker
0
_10
18 23 28 33 38 43 48 53 58 63 68
Time (sec)
Figure 1
Electrophoretic separation of different plant total RNA using the RNA 6000 Nano Kit with the Plant RNA assay.
Representative electropherograms indicate the position of 18S and 25S rRNA peaks. Unlabeled peaks correspond to
additional ribosomal RNA. RIN values are shown.
2
Inter chip (intra-day, n = 15)
Inter chip (inter-day, n = 30)
Mean RIN
SD
%CV
Mean RIN
SD
%CV
Arabidopsis
7.29
0.13
1.78
7.31
0.15
2.05
Lettuce
7.61
0.19
2.49
7.76
0.12
1.54
Sunflower (root)
8.02
0.29
3.61
8.41
0.46
5.46
Sunflower (leaf)
6.88
0.13
1.88
6.87
0.12
1.74
Tomato
6.48
0.12
1.85
6.51
0.12
1.84
Tobacco
6.75
0.16
2.37
6.78
0.15
2.21
Table 2
Precision of plant RNA assay method.
Fluorescence
15
25S
25
0 min
RIN = 6.7
18S
20
15 Marker
10
5
10
18S
25S
5 min
RIN = 6.5
5
0
20
20
15 Marker
18S
25S
10
10 min
RIN = 5.9
5
Fluorescence
_5
18 23 28 33 38 43 48 53 58 63 68
Time (sec)
15 Marker
20 min
RIN = 4.7
10
18S
5
25S
0
_5
18 23 28 33 38 43 48 53 58 63 68
Time (sec)
_5
18 23 28 33 38 43 48 53 58 63 68
Time (sec)
30
25
Fluorescence
The Agilent 2100 Bioanalyzer System
Plant RNA assay allows rapid,
automated analysis of plant RNA
samples with excellent precision. The
RIN algorithm of the Plant RNA assay
provides a user-independent
assessment of total plant RNA
integrity.
Marker
0
_5
18 23 28 33 38 43 48 53 58 63 68
Time (sec)
0
Conclusion
Fluorescence
30
Fluorescence
Time-dependent degradation of
plant RNAs
RNA is sensitive to degradation
with many factors (cellular RNases,
temperature or chemical treatment)
contributing to this process. To mimic
a natural degradation process, tobacco
samples were incubated at room
temperature over 0 to 40 minutes. The
degree of degradation was monitored
through analysis on the Agilent 2100
Bioanalyzer System and RNA Nano
6000 kit. Figure 2 shows representative
tobacco RNA electropherograms at
different time points. As expected, the
RIN decreases as degradation
progresses. The Agilent 2100
Bioanalyzer System profiles show
minimal change in 25S/18S rRNA ratio
(data not shown) or RIN at time points 0
and 5 minutes. With longer incubations,
the RNA size distribution shifts toward
smaller fragments result-ing in
decreased RINs (Figures 2 and 3). As
expected, the samples showed the
highest degree of degradation with a
mean RIN value of 3.6 after 40 minutes
at room temperature.
20
15
40 min
RIN = 3.8
Marker
18S
10
25S
5
0
_5
18 23 28 33 38 43 48 53 58 63 68
Time (sec)
Figure 2
Time course degradation of tobacco RNA. Total RNA samples were incubated at room temperature at selected time
points and separated with RNA Nano 6000 kit. Electropherograms indicate the position of 18S and 25S rRNA peaks.
RIN values of individual samples are shown.
3
References
2.
A.Schroeder, O. Mueller, S. Stocker,
R. Salowsky, M. Leiber, M. Gassmann,
S. Lightfoot, W. Menzel, M. Granzow
and T. Ragg , “The RIN: an RNA
integrity number for assigning integrity
values to RNA measurements.” BMC
Mol Biol., 7:3, 2006.
8
6
RIN
1.
H. Daniell, S. Chebolu, S. Kumar, M.
Singleton, R. Falconer, “Chloroplastderived vaccine antigens and other
therapeutic proteins.” Vaccine.,
23:1779–1783, 2005.
4
2
0
-10
0
10
20
30
40
50
Degradation time (mins)
Figure 3
Assessment of plant RNA degradation. Different degradative states of tobacco RNA samples were analyzed with the
Plant RNA assay and RNA 6000 Nano kit (average of forty seven measurements, data represent mean ± SD).
www.agilent.com/genomics/RNA
For Research Use Only.
Not for use in diagnostic procedures.
© Agilent Technologies, Inc., 2011, 2016
Published in USA, January 21, 2016
Publication Number 5990-8850EN